Hormone-refractory prostate cancer is a major health threat to American males. Modulation of apoptotic responses represents a hopeful weapon against the disease. Our laboratory has been studying the molecular mechanisms of prostate cancer cell apoptosis and is involved in apoptosis-based anti-prostate cancer drug development. We recently have discovered a novel apoptotic signaling pathway in prostate cancer cells which is dependent on the mitochondrial respiratory chain (MRC), initiated and executed in the mitochondria in situ, and then propagated and amplified in the cytosol. The main aim of this proposal is to elucidate this novel apoptotic pathway at the molecular level. The long-term goal of this project is to understand the role of mitochondria in regulating prostate cancer cell survival, apoptosis, and sensitivity to chemotherapeutic drugs and gamma-radiation and to generate scientific data to help design more specific and more efficacious anti-prostate cancer drugs targeted to mitochondria by triggering apoptotic responses. This mitochondrial-signaled apoptotic pathway appears to involve the following sequential molecular events: 1) abnormal activation of MRC by apoptotic stimuli; 2) increased transport of cytochrome c from cytosol (either from pre-existing cytosolic pool or from enhanced gene transcription) to the mitochondria; 3) activation of procaspase-3 in the mitochondria; 4) collapse of MRC, mitochondrial permeability transition, and subsequent cytochrome c release from mitochondria leading to the activation of cytosolic procaspases; and 5) eventual cell demise. The key feature of this pathway is procaspase-3 activation in mitochondria in situ, a process dependent on the MRC upregulation and cytochrome c enrichment into the organelle which all precede cytochrome c release from mitochondria to the cytosol. We term this novel pathway MADAP for mitochondria activation-dependent apoptotic pathway. The current proposal attempts to decipher the MADAP with the following specific aims: 1) To elucidate the molecular mechanisms and the role of cytochrome c translocation to mitochondria in MADAP; 2) To elucidate how mitochondrial procaspase-3 is activated in the organelle and the role of mitochondrial caspase- 3 in MADAP; and 3) To determine the role of some Bc1-2 family proteins in regulating MADAP. The above goals will be accomplished by a combination of cell, biochemical, and molecular biology approaches including utilization of respiration-deficient cells, confocal microscopy, quantitative immunogold electron microscopy, construction of HA-, FLAG-, and GFP-tagged expression vectors, microinjection, in vitro reconstitution, DNA transfection and multiple standard molecular biology techniques (Northern, Western, immunoprecipitation, immunodepletion, pulse- chase, PCR, cDNA library, etc). The significance of this project is: 1) to advance our understanding of the role of mitochondria in general and mitochondrial caspase(s) in particular in apoptosis; 2) to elucidate the crucial role of mitochondria in regulating prostate cancer cell apoptosis; and 3) to lay a scientific foundation for the development of novel anti-prostate cancer therapeutics.